There is growing interest in the use of lentiviral vectors, particularly for cancer immunotherapy and the treatment of monogenic diseases. Manufacturing of these vectors is challenging primarily due to cytotoxic effects of vector components resulting in low cell culture titres and vector instability leading to low purification yields. In addition, currently used processes are typically not scalable as they rely on adherently cultured cells and unit operations such as batch centrifugation and gel filtration. To improve process scalability, suspension adaptation of a lentiviral vector packaging cell line was attempted, however, cell aggregation could not be prevented. For vector clarification it was found that membranes with pore sizes of 0.22 µm resulted in recoveries less than 50%, whereas the use of 0.45 µm membranes resulted in recoveries close to 100%. Successful vector concentration utilising centrifugal filters was possible with a membrane molecular weight cut-off (MWCO) of 100 kDa, whereas a 300 kDa MWCO led to low recoveries. Chromatography stationary phases that allow convective mass transfer, such as membranes and monoliths, are becoming increasingly popular for purification of large molecules. Lentiviral vector was found to bind monoliths with weak and strong anion exchangers over a wide range of conditions. Vector elution conditions determined for membrane- and monolith-based resins in high-throughput 96-well plate format were found to not be indicative of gradient elution conditions for 1 mL versions of these resins operated by a chromatography system. The thermolability of lentiviral vectors leads to a requirement for storage at less than -65°C. Inexpensive mixtures of sugars in combination with a glycine-derivative were studied for their ability to stabilise a lentiviral vector during freeze-drying and subsequent thermochallenge. The amorphous solid formed upon freeze-drying was able to stabilise the vector for up to 12 weeks at 4°C and eight weeks at 25°C. This is in comparison to formulation in phosphate buffered saline, where more than 90% of infectious titre was lost immediately upon freeze-drying.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:763100 |
| Date | January 2016 |
| Creators | Nilsson, S. M. |
| Contributors | Mukhopadhyay, T. |
| Publisher | University College London (University of London) |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://discovery.ucl.ac.uk/1485725/ |
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